Oxidative stress plays a major role in the pathogenesis of many human diseases, and in particular, neurodegenerative diseases. Treatment with antioxidants, which may reduce particular free radical species, therefore, may prevent tissue damage and improve both survival and neurological outcome. Hydrogen peroxide is a by-product of many important cellular processes; however, it is also a known generator or precursor of free radicals in physiological environments. Catalase is a naturally occurring antioxidant enzyme (redox protein) that prevents excessive buildup of hydrogen peroxide by catalyzing the breakdown of hydrogen peroxide into water and oxygen. Antioxidant drugs with activity that mimics the cellular enzyme catalase (i.e. catalase-like activity) may slow the progression of various oxidative stress related diseases and events, such as ischemic stroke.
The origin of the use of nanoceria in nanomedicine can be traced to the seminal work of Bailey and Rzigalinski, wherein the application of ultrafine cerium oxide particles to brain cells in culture was observed to greatly enhanced cell survivability, as described by Rzigalinski in Nanoparticles and Cell Longevity, Technology in Cancer Research & Treatment 4(6), 651-659 (2005). More particularly, rat brain cell cultures in vitro were shown to survive approximately 3-4 times longer when treated with 2-10 nanometer (nm) sized cerium oxide nanoparticles synthesized by a reverse micelle micro emulsion technique, as disclosed by Rzigalinski et al. in U.S. Pat. No. 7,534,453, filed Sep. 4, 2003.
Subsequently, a host of problems with these particular nanoceria particles was disclosed by Rzigalinski et al. in WO 2007/002662. Nanoceria produced by the reverse micelle micro emulsion technique suffered as follows: (1) particle size was not well-controlled within the reported 2-10 nanometer (nm) range, making variability between batches high; (2) tailing of surfactants, such as sodium bis(ethylhexyl)sulphosuccinate, also known as docusate sodium or (AOT), used in the reverse micelle synthetic process into the final product caused toxic responses; (3) inability to control the amount of surfactant tailing posed problems with agglomeration when these nanoparticles were placed in biological media, resulting in reduced efficacy and deliverability; and (4) instability of the valence state of cerium (+3/+4) over time. Thus, the cerium oxide nanoparticles produced by the reverse micelle micro emulsion technique were highly variable from batch to batch, and showed higher than desired toxicity to mammalian cells.
As an alternative, Rzigalinski et al. in WO 2007/002662 describe the biological efficacy of nanoceria synthesized by high temperature techniques, obtained from at least three commercial sources. These alternative sources of cerium oxide nanoparticles were reported to provide superior reproducibility of activity from batch to batch. It was further reported that, regardless of source, cerium oxide particles having a small size, narrow size distribution, and low agglomeration rate are most advantageous.
These inventors (Rzigalinski et al.) also report therein that for delivery, the nanoparticles were advantageously in a non-agglomerated form. To accomplish this, they reported that stock solutions of about 10% by weight of nanoceria could be sonicated in ultra-high purity water or in normal saline prepared with ultra-high purity water. However, we have confirmed what others have observed, that sonicated aqueous dispersions of nanoceria (synthesized by high temperature techniques and obtained from commercial sources) are highly unstable, and settle rapidly (i.e. within minutes), causing substantial variability in administering aqueous dispersions of nanoceria derived from these sources. We have also observed that administration of these sonicated aqueous dispersions of nanoceria (e.g. obtained from Sigma-Aldrich) to mice result is rapid deposition of ceria in the liver and kidneys along with rapid decline in the health of the animals.
While cerium oxide containing nanoparticles can be prepared by a variety of techniques known in the art, the particles typically require a stabilizer to prevent undesirable agglomeration. In regard to biocompatible nanoceria stabilizers used previously, Masui et al., J. Mater. Sci. Lett. 21, 489-491 (2002) describe a two-step hydrothermal process that directly produces stable aqueous dispersions of ceria nanoparticles that use citrate buffer as a stabilizer. However, this process is both time and equipment intensive, requiring two separate 24 hours reaction steps in closed reactors.
DiFrancesco et al. in PCT/US2007/077545, METHOD OF PREPARING CERIUM DIOXIDE NANOPARTICLES, filed Sep. 4, 2007, describes the oxidation of cerous ion by hydrogen peroxide under highly acidic conditions (pH<4.5) in the presence of biocompatible α-hydroxy carboxylic acid stabilizers, such as lactic acid, tartaric acid, gluconic acid and 2-hydroxybutanoic acid. Specifically, the stabilizer lactic acid and the stabilizer combination of lactic acid and ethylenediaminetetraacetic acid (EDTA) are shown in working examples to directly produce stable dispersions of nanoceria particles of average particle size in the range of 3-8 nm under highly acidic reaction conditions.
Commonly assigned U.S. Pat. No. 9,034,392 describes the oxidation of cerous ion by hydrogen peroxide in the presence of citric acid (CA) and ethylediaminetetraacetic acid (EDTA), wherein the molar ratio of CA to EDTA ranges from about 0.1 to about 3.0, whereby aqueous dispersions of stabilized cerium oxide nanoparticles are formed directly in the reaction mixture, without isolation of the nanoparticles. Cerium oxide nanoparticles stabilized with a combination of CA and EDTA in this range of molar amounts are shown to synergistically reduce cell death due to oxidative stress in an ex vivo murine brain slice model of ischemic stroke.
Commonly assigned US Patent Application 2014/0322333 describes the oxidation of cerous ion by hydrogen peroxide in the presence of citric acid and a chelant selected from the group consisting of nitrilotriacetic acid (NTA), ethylene glycol tetraacetic acid (EGTA) and diethylenetriaminepentaacetic acid (DTPA), whereby aqueous dispersions of stabilized cerium oxide nanoparticles are formed directly in the reaction mixture, without isolation of the nanoparticles. These stabilized cerium oxide nanoparticles are shown to reduce cell death due to oxidative stress in an ex vivo murine brain slice model of ischemic stroke.
As described above, various methods have been reported for preparing aqueous dispersions of biocompatibly stabilized cerium oxide nanoparticles. Antioxidant drugs with increased catalase-like activity will more quickly reduce the tissue damage resulting from an excess buildup of hydrogen peroxide, and/or may enable a corresponding reduction in drug dosage, thereby reducing the drug cost and/or drug side effects to the patient. Thus, there remains a need for increased catalase-like activity in aqueous dispersions of stabilized cerium oxide nanoparticles.